Copper on is an essential metal ion found in various organs and plays crucial roles in living systems as cofactors of cytoplasmic enzymes, mitochondrial enzymes and membrane-bound oxidases in production of cellular energy, reduction of oxygen molecules, signal transduction, or the like. Copper(I) ion (Cu+) in reduced state is internalized in cells but copper(II) ion (Cu2+) in oxidized state is often found in cells under oxidative environment. Abnormality in the regulation of copper ion level may cause severe diseases such as Menkes disease, Wilson's disease, Alzheimer's disease and prion disease and increased copper ion level in the body may cause gastrointestinal disorders, damage to the liver and kidneys, and so forth (Gaggelli, E.; Kozlowski, H.; Valensin, D.; Valensin, G. Chem. Rev. 2006, 106, 1995; Macreadie, I. G. Eur. Biophys. J. Biophy. 2008, 37, 295; Georgopoulos, P. G.; Roy, A.; Yonone-Lioy, M. J.; Opiekun, R. E.; Loy, P. J. J. Toxicol. Env. Heal. B 2001, 4, 341).
Total-reflection X-ray fluorescence (TRXRF) and atomic absorption spectroscopy are available as methods for quantitatively measuring copper on in normal and abnormal tissues. However, these methods are incapable of differentiating the oxidation state of copper and are not suitable for quantitative measurement of copper on level in vivo due to low detection sensitivity (Kucharzewski, M.; Braziewicz, J.; Majewska, U.; Gozdz, S. Biol. Trace Elem. Res. 2003, 92, 1).
Two-photon microscopy can solve the above problems and allows quantitative measurement of metal ions in vivo. The two-photon microscopy is a technique that uses two near-infrared photons with energy lower than that of confocal microscopy for excitation and allows imaging up to a very high depth inside cells (Helmchen, F.; Denk, W. Nat. Methods 2005, 2, 932; Zipfel, W. R.; Williams, R. M.; Webb, W. W. Nat. Blotechnol. 2003, 2, 1369; Kim, H. M.; Cho, B. R. Acc. Chem. Res. 2009, 42, 863; Kim, H. M.; Cho, B. R. Chem. Asian J. 2011, 6, 58).
Korean Patent Publication No. 2013-0039680 discloses a two-photon fluorescent dye selective for copper(I) ion and a manufacturing method thereof. Korean Patent Registration No. 886,722 discloses a two-photon dye capable of monitoring magnesium in the cytoplasm in real time and a monitoring method using the same. And, Korean Patent Registration No. 976,623 discloses a two-photon dye capable of monitoring calcium in cells in real time. Although various two-photon probes for detecting metal ions in vivo have been developed, development of a two-photon probe having selectivity for copper(II) on has not been reported. Accordingly, development of a two-photon probe capable of selectively detecting and quantitating the biologically important copper(II) on is needed.